School of Medicine, Ningbo University, Ningbo, People's Republic of China ; Department of Gynecological Oncology, Ningbo Women and Children's Hospital, Ningbo, People's Republic of China.
Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, People's Republic of China.
Onco Targets Ther. 2013 Nov 25;6:1707-17. doi: 10.2147/OTT.S54217. eCollection 2013.
The present study aims to investigate the subunit expression and enzyme activity of ribonucleotide reductase in cervical cancer patients, and detect the combined effect of the ribonucleotide reductase inhibitor gemcitabine and the chemotherapeutic agent carboplatin on cervical cancer cell lines.
Using quantitative reverse transcription polymerase chain reaction, Western blotting, and cytidine 5'-diphosphate reduction assays, we tested the expression and activity of ribonucleotide reductase in cervical cancer patients. The antitumor activity of gemcitabine and/or carboplatin treatments to SiHa and CaSki human cervical cancer cell lines were assessed by Cell Counting Kit-8 viability assay, EdU incorporation assay, immunofluorescence assay, flow cytometry assay, and Western blotting methods. Additionally, synergistic efficacy was quantitatively analyzed using a combination index based on the Chou-Talalay method.
The mRNA levels of three ribonucleotide reductase subunits were all upregulated in the cervical cancer tissues compared with normal tissues (P<0.0001). Consistently, the protein expression and enzyme activity of ribonucleotide reductase were also increased in the cervical cancer tissues. Interestingly, gemcitabine inhibited DNA synthesis and carboplatin induced DNA damage. Further, the combined drug regime had a significant synergistic effect on inhibiting cervical cancer cell viability (log10[combination index] <0) via enhanced DNA damage and cell apoptosis.
The expression and activity of ribonucleotide reductase was increased in cervical cancer. Our study demonstrated the synergistic cytotoxicity of gemcitabine and carboplatin, through inhibiting DNA synthesis and increasing cell apoptosis in cervical cancer cell lines. This evidence might provide a rational clue of their combined application to improve cervical cancer treatment.
本研究旨在探讨宫颈癌患者核苷酸还原酶亚基表达和酶活性，并检测核苷酸还原酶抑制剂吉西他滨与化疗药物卡铂联合对宫颈癌细胞系的作用。
采用实时定量逆转录聚合酶链反应、Western blot 及胞嘧啶 5'-二磷酸还原实验检测宫颈癌患者核苷酸还原酶的表达和活性。采用细胞计数试剂盒-8 活力检测、EdU 掺入实验、免疫荧光实验、流式细胞术和 Western blot 方法评估吉西他滨和/或卡铂对 SiHa 和 CaSki 人宫颈癌细胞系的抗肿瘤活性。此外，还采用 Chou-Talalay 方法基于组合指数定量分析协同作用。
与正常组织相比，宫颈癌组织中三种核苷酸还原酶亚基的 mRNA 水平均上调（P<0.0001）。同样，宫颈癌组织中核苷酸还原酶的蛋白表达和酶活性也增加。有趣的是，吉西他滨抑制 DNA 合成，卡铂诱导 DNA 损伤。此外，联合药物治疗通过增强 DNA 损伤和细胞凋亡对抑制宫颈癌细胞活力具有显著协同作用（log10[组合指数] <0）。
宫颈癌中核苷酸还原酶的表达和活性增加。本研究表明，吉西他滨和卡铂联合具有协同细胞毒性作用，通过抑制 DNA 合成和增加宫颈癌细胞系中的细胞凋亡。这一证据可能为它们联合应用以改善宫颈癌治疗提供合理线索。
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